HPV18E6基因沉默对宫颈癌Hela细胞生长和凋亡的影响

siRNA targeting of HPV18E6 inhibits the growth of Hela cells and induces apoptosis

目的:观察RNA干扰法沉默HPV18E6基因表达对宫颈癌Hela细胞生长和凋亡的影响,探索宫颈癌基因治疗的新途径。方法:针对HPV18E6 mRNA序列合成一对60bp的编码siRNA的DNA模板和一对60bp的非特异性对照DNA模板,构建pSUPER-siRNA和pSUPER-con重组质粒,瞬时转染Hela细胞;采用RT-PCR法检测质粒转染后细胞HPV18E6基因表达的变化,用蛋白免疫印迹法检测转染后Hela细胞p53、p21、Bcl-2和Bax蛋白表达变化,以细胞计数法检测细胞生长情况,Hoechest/PI双荧光活细胞染色法检测细胞凋亡。结果:pSUPER-siRNA质粒转染能有效降低HPV18E6在mRNA水平的表达,转染后48小时,抑制效率达70%以上;转染后细胞p53、p21和Bax蛋白表达显著增加,Bcl-2蛋白表达减少。RNA干扰法沉默HPV18E6基因表达后,Hela细胞增殖受到明显抑制,细胞凋亡率明显增加。结论:pSUPER-siRNA质粒转染可有效抑制HPV18E6在人宫颈癌Hela细胞中的表达,抑制Hela细胞生长并促进其凋亡。以HPV18E6为靶点的RNA干扰技术可望成为宫颈癌基因治疗的新途径。

Objective： To study the effects of HPVI8E6 -targeting siRNA on the growth and apoptosis of Hela cells and to explore a new pathway for cervical cancer genetherapy . Methods：A pair of DNA template coding siRNA against HPV18E6 and itg control template were synthesized to reconstruct pSUPER - siRNA and pSUPER - control plasmids,which was transfected transiently into Hela cells. The HPV18E6 expression in Hela cells was detected by RT- PCR. The protein expression of p53 ,p21, Bcl -2 and Bax was detected by Western blot. The proliferation and apoptosis of Hela cells was detected respectively by cell counting and Hoechest/PI fluorescent vital staining. Results ： The siRNA expression vector against HPV18E6 mRNA was successfully constructed. The transfection of pSUPER - siRNA targeting HPV18E6 effectively decreased the expression of HPV18E6 in the Hela cells and the decrease rate was more than 70% in 48 hour after transfection.. In the Hela cells transfected with pSUPER - siRNA plasmid, the protein expression of p53, p21 and Bax was increased significantly while the expression of Bcl - 2 was decreased markedly. After silence of HPV18E6 gene, the proliferation of Hela cells was obviously inhibited and the apoptotic rates were significantly increased. Conclusion： HPV18E6 -targeting siRNA can significantly inhibit HPV18E6 expression, suppress the growth of Hela cells and induce apotosis. The RNA interfering technique targeting of HPV18E6 maybe can provide a new pathway for the gene therapy of cervical carcinoma.